ES2356678T3 - BIOMATRIZ OF COLLAGEN AND METHOD TO PRODUCE. - Google Patents

Abstract

Method of preparing a collagen biomatrix, which comprises the following steps: preparing collagen fibers from rat tails; transfer the collagen fibers to an acetic acid solution with a pH value of 3.0 to 4.0; incubate the collagen fibers in the acetic acid solution at a temperature of 2 to 10 ° C for a period of 3 to 14 days; separate the undissolved collagen particles, whereby a collagen solution is obtained whose collagen content is 3 to 8 mg / ml; and mix the collagen solution obtained with a buffer solution of 7.5 to 8.5 pH at a temperature of 2 ° C to 10 ° C and increase the temperature to achieve gelation, thereby obtaining a biomatrix with 1.5 to 4.0 mg of collagen per 1 ml of biomatrix and a pH value of 7.0 to 7.8.

Description

The present invention relates to a biomatrix and methods for its preparation.

Joint diseases are very common disorders, which are accompanied by a large number of discomforts for affected people. Thus, for example, bone and cartilaginous defects play an important role in the pathogenesis of osteoarthritis and also in post-traumatic states and in dislocated stents. These defects can be treated with the use of autologous or homologous bone or cartilage, or by suitable substitute materials. Since autologous materials are not available unlimitedly, in the case of homologous grafts, infection aspects must be taken into account.

The main problem of degenerative or traumatic joint disorders is the poor regeneration capacity of damaged cartilage. A known treatment of these cartilaginous defects is the autologous chondrocyte implant (ACT) as a method of biological therapy. With this therapeutic method it was possible to detect for the first time the new formation of hyaline cartilage in a joint cartilaginous defect.

The principle of this method is based on injecting the patient's cartilaginous cells, grown in vitro after debriding the degenerated cartilage, under a periodic flap sewn over the defect (Peterson method). The procedure is technically complicated and poses methodological problems; For example, it does not guarantee that cartilaginous cells remain long in the crack. Furthermore, it is not clear that cultured cartilaginous cells are capable of forming the matrix necessary to permanently fill the crack at the time of transplantation. The transplanted cells are mostly in a dedifferentiated state and have morphological and physiological similarities with fibroblasts. twenty

The use of new materials is known from DE 197 21 661 A1, to replace bones or cartilage, which, although known, are characterized by having a specific structure. It is a bone or cartilage implant based on a three-dimensional network consisting basically of a large number of rods of a totally or partially bioabsorbable material, arranged regularly. The rods form a three-dimensional geometric structure whose elasticity and resistance are adapted to the tissue that must be replaced in the patient's body. The rods can be made of poly-D-lactides, poly-L-lactides, poly-DL-lactides, hydroxyapatites, calcium phosphates or mixtures of these substances that essentially contain calcium phosphates or hydroxyapatites, collagen, agar or gelatin.

WO 99/08728 discloses a mixture of osteoinductive or chondroinductive factors included in nanospheres. It also reveals a system formed by a biodegradable matrix containing, for example, type I or type II collagen in which the factors involved by the nanospheres are included. The nanospheres are made up of polymeric particles.

From WO 95/33821 it is known to make three-dimensional collagen structures whose interstices are bridged, eg by fibroblasts or chondrocytes, and this lattice is grown in a culture medium. This document describes the culture of these cells on a three-dimensional grid, as well as the use of this artificial tissue as a cartilaginous prosthesis. 35

WO 98/17791 describes the extraction and use of chondrocyte precursors, which can be methodically multiplied by culture and used as therapeutic cartilaginous tissue. It also describes the culture of chondrocytes on a three-dimensional grid whose interstices are bridged by the chondrocytes.

WO 99/00152 discloses a method for producing a bioartificial graft, which consists in separating all the antigen-reactive cells from an allogenous or xenogenic tissue by enzymatic or chemical treatment and colonizing with the desired autologous cells the cell-free material. and not denatured thus obtained, resulting in a graft immediately ready to transplant.

The disadvantage of the known methods is that the transplanted or cultured cartilaginous cells do not resume their original synthesizing capacity.

The cartilaginous cells included in the intracellular substance of the cartilage - the chondrocytes - dedifferentiate precisely during the culture period under in vitro conditions. While primary cartilaginous cells (P0 culture) still show specific abilities to synthesize cartilaginous cells after their isolation from cartilaginous tissue, as demonstrated by the formation of type II, IX and XI collagen, cultured cells lose these typical characteristics during successive passages in vitro (P1-PX).

Also, unlike other cells, cartilaginous cells are difficult to grow. Cultured cartilaginous cells are in a dedifferentiated state and morphologically and physiologically resemble fibroblasts. However, especially in case of large defects, multiplication by culture of autologous chondrocytes and their passage is necessary to produce adequate amounts of transplant material from small samples.

Until now, dedifferentiation of cartilaginous cells can only be avoided by adding additional growth stimulants, which makes it difficult to use these cultures in vivo, as growth stimulants can interact unfavorably with the immune system of the recipient organism. In addition, known methods do not ensure that cells maintain their natural or almost natural metabolism during culture, even after several passages. 5

With current methods, the three-dimensional structures known to grow cartilaginous cells cannot also be specifically adapted to the corresponding cartilage defect. Nor is it guaranteed that the transplanted cartilaginous cells remain in a lasting and differentiated manner at the site of the defect, allowing cartilage regeneration.

No transplant method is really known to date capable of ensuring that the transplanted cartilaginous cells remain permanently in the place of the defect, thus ensuring the corresponding regeneration of the cartilage.

Therefore the technical problem object of the present invention is to provide cartilaginous grafts, as well as methods and means for their production, which allow a better treatment of the conditions of the cartilage, especially a better cure or regeneration of the treated cartilaginous defect. fifteen

The present invention solves this problem by offering means for redifferentiating and / or multiplying dedifferentiated cartilaginous cells, based on culturing the dedifferentiated cartilaginous cells in a three-dimensional gelatinous biomatrix where they can redifferentiate and resume their specific cellular metabolic activity.

The biomatrix according to the present invention contains a newly formed collagen framework from a collagen solution, preferably fresh, with a minimum concentration of 1.5 mg of collagen / ml of biomatrix, preferably 1.5 to 4 mg of collagen / ml of biomatrix. This collagen framework is obtained from an acid solution of collagen preferably free of cells, preferably with a pH value of 0.1 to 6.9, preferably 2.0 to 5.0, especially 3.0 to 4.5, specifically from 3.2 to 4.2 and with a special preference of 3.8. Said collagen solution is prepared and stored between 2 and 10 ° C, preferably at 4 ° C. To prepare a cell-free biomatrix, this collagen solution is mixed between 2 and 10 ° C, preferably at 4 ° C, with a solution of medium, specifically a cell culture medium, a buffer, for example HEPES, and serum, especially autologous human serum, and gels by increasing the temperature, for example, to room temperature or 37 ° C. To prepare a biomatrix containing cells, preferably precultured cells, for example cartilaginous cells or precursors of cartilaginous cells, are introduced into the solution of medium, buffer and serum, which is then mixed between 2 and 10 ° C, preferably at 4 ° C, with the solution of collagen equally tempered between 2 and 10 ° C, preferably at 4 ° C. Cells thus included in the biomatrix can be subsequently cultured and, if necessary, removed by dissolution. It is then gelled for example at room temperature or at 37 ° C and the biomatrix is coated with cell culture medium. The method of the present invention allows advantageously to perform an intermediate culture of the cells in a biomatrix according to the present invention, so that the chondrocytes, for example of the P2 culture, are stimulated in the three-dimensional biomatrix to synthesize again typically cellular cellular matrix proteins. , especially collagen II, while no known collagen formation can be detected in known cultures. Then, advantageously, the cells can be extracted again from the biomatrix and cultured. Thanks to the present invention, redifferentiation of dedifferentiated cartilaginous cells allows culturing and / or multiplying chondrocytes for a long period of time, without the cells losing the specific synthesis capacity necessary for cartilage formation. Therefore, advantageously, starting even with a small amount of cartilaginous tissue, sufficient cellular material can be available for the production of cartilage grafts.

With respect to the present invention, the term "cell culture" refers to a maintenance, especially in vitro, of the vital functions of cells in a suitable environment - for example, with input and extraction of metabolic products and educts - and also specifically a cell multiplication.

With respect to the present invention, cartilaginous cells or chondrocytes are understood to mean cartilaginous cells of natural origin or modified by genetic engineering, or their precursors, which can be of animal or human origin.

The culture of differentiated cartilaginous cells is foreseen, especially maintaining their differentiation, in a three-dimensional biomatrix of the aforementioned characteristics. The metabolic activities of the primary culture are advantageously preserved, without dedifferentiation of the differentiated cartilaginous cells. The present invention also provides a method of chondrocyte culture comprising a suppression of redifferentiation.

It is envisaged that the cartilaginous cells, subject to the control of their function, their morphology and / or their state of differentiation, are introduced into the aforementioned three-dimensional biomatrix, cultured and checked simultaneously and / or subsequently. Thus, for example, the metabolism of cartilaginous cells can be analyzed advantageously in vitro, before using them in vivo. The analysis can be, for example, the measurement of metabolic activities, as well as a morphological or functional test.

The present invention also relates to screening and diagnostic methods, which consist of culturing cells.

cartilaginous according to the procedure described above and analyze them simultaneously or subsequently, to determine, for example, their physiological, morphological and / or biomolecular parameters. In this way, degenerative or traumatic conditions of the joints or their absence can be specifically detected. Within the framework of these methods, the effects of potential drugs and / or pathogens, antigens or the like on cultured cells can also be examined, for example in drug selection processes. According to a preferred embodiment, the cells are cultured in the presence and absence of the investigated agent, comparing the observed effects with each other.

In an advantageous embodiment, the cartilaginous cells are extracted after culturing them in the three-dimensional biomatrix, for example by treatment with collagenase and subsequent concentration, and then continued to be cultured in a current two-dimensional or three-dimensional cell culture. Thus, the advantages of two-dimensional and three-dimensional culture can be combined favorably.

In a particularly advantageous embodiment, the cartilaginous cells are introduced into a three-dimensional biomatrix according to the present invention and cultured therein, so that a transplantable cartilaginous prosthesis, also called cartilage graft, can then be obtained. In a preferred embodiment, this cartilaginous prosthesis may be an articular cartilage prosthesis. Thus, for example, the biomatrix can already be advantageously adapted to the shape of the cartilaginous defect during the cell culture phase.

An advantageous embodiment is a previously cited method for making a cartilaginous prosthesis, especially articular, according to which the cartilaginous cells are preferably extracted from the three-dimensional biomatrix after culturing therein, preferably by treatment with collagenase and centrifugal separation, and they are still cultivated, preferably at high cell density, in a current two-dimensional or three-dimensional culture, whereby a transplantable, especially articular, cartilaginous prosthesis can be obtained.

The present invention also relates to a cartilaginous, especially articular, prosthesis elaborated by the method of the present invention and by a conventional subsequent or / and previous conventional culture method.

The present invention also relates to a biomatrix, preferably gelatinous, in which said culture methods can be performed, that is to say a biomatrix both without cartilage cells and with them. In the latter case, the combination of biomatrix and differentiated or redifferentiated cartilaginous cells cultured therein is also called the cell-matrix-cartilage or biograft system. This biograft can be used directly in the therapy of diseases or defects of the cartilage.

According to the present invention, a biomatrix means a gelatinous structure that brings collagen, cell culture medium, serum and buffer, especially Hepes buffer. The collagen solution used to prepare the biomatrix is a solution that carries a large content of native non-denatured collagen in aqueous acid medium, specifically with a pH value of 0.1 to 6.9, preferably 2.0 to 5.0 , especially from 3.0 to 4.5, in particular from 3.2 to 4.2 and with particular preference of 3.8, for example in acetic acid, in particular 0.1% acetic acid solution. The high content refers to a part of non-denatured collagen on total collagen in solution  50%, especially  60,  70,  80,  90 or  95, especially  99%. In a preferred embodiment, no lyophilized collagen is used. The collagen content in the solution is preferably between 3 mg of collagen / ml of solution and 8 mg of collagen / ml of solution, preferably 6 mg of collagen / ml of solution. In a preferred embodiment, a collagen is used which, once isolated from rat tails, for example, has been incubated in 0.1% acetic acid for 3 to 14 days at 4 ° C and under stirring, separating the centrifuges by centrifugation. undissolved collagen fractions. DMEM (Eagle medium modified by Dulbecco) is advantageously used as a cell culture medium, but any other cell culture medium that allows culturing cartilaginous cells can also be used. As the serum, in the preferred embodiment autologous human serum is used and as a Hepes buffer, for example. In the preferred embodiment, the concentration of Hepes in the solution is adjusted to 3 M. In the preferred embodiment, the pH value of the solution of cell culture medium, buffer and serum is 7.5 to 8.5. , for example 7.6 to 8.2, especially 7.8. Obviously the biomatrix may also contain other factors, for example growth factors, adhesive agents, antibiotics, selection agents or the like.

Therefore, the present invention also relates to methods for making a biomatrix, according to which, in a first stage, fresh collagen is prepared from rat tails, for example, by collecting in buffer solution collagen fibers extracted from the tissue that It contains, disinfecting them superficially in alcohol, then washing them 50 in buffer solution and then transferring them to an acid solution whose pH value is 0.1 to 6.9, preferably 2.0 to 5.0, especially 3.0 to 4.0, specifically 3.3, for example a 0.1% acetic acid solution. Then, at another stage, the dissolved collagen is stirred between 2 and 10 ° C, especially at 4 ° C, for a few days, for example 3 to 14 days, the undissolved collagen particles are separated by centrifugation and stored between 2 and 10 ° C, for example at 4 ° C, a finished solution containing 3 mg / ml up to 8 mg / ml collagen. Of course the solution can also be kept frozen, for example between -10 ° C and -80 ° C, especially -20 ° C. In a third stage this collagen solution is preferably mixed in a 1: 1 ratio with a solution of pH 7.5 to 8.5, preferably 7.6 to 8.2, especially 7.8, which contains cell culture medium doubly concentrated, serum and buffer, obtaining a biomatrix having a pH value of 7.0 to 7.8, preferably 7.4. To prepare a biograft, the solution of the doubly concentrated cell culture medium, serum and buffer is mixed with 60

Precultured and centrifuged cartilaginous cells, preferably using 2 x 104 to 2 x 107 cells per ml, especially 2 x 106 cells. Then, this solution of pH 7.5 to 8.5, preferably 7.6 to 8.2, especially 7.8, is mixed between 2 and 10 ° C, in particular at 4 ° C, with the collagen solution mentioned above in 1: 1 ratio Then, the gel solution is pipetted into culture vessels and after gelation at 37 ° C it is coated with medium. Then the biograft is grown for 3 to 8 days, so that it is available for transplants. 5

The biograft arrives in the operating room in culture vessels and then the doctor can adapt it by mechanical work to the size, thickness and shape of the defect, for example with a scalpel. The biograft is then fixed in the crack, for example with tissue adhesive, specifically with fibrin adhesive. After about 5 minutes the biograft is firmly anchored in the crack. The operation can also be performed by arthroscopy, since the biograft is flexible. 10

Another use of the biograft is a combination of the biomatrix of the present invention with bones. In this case, the biograft is applied, for example, on autologous bone cylinders of the iliac crest, for example with fibrin adhesive. The present invention makes it possible to treat especially osteochondritis dissecans and cracks of the head of the femur due to dislocation of the hip by cerebral paresis, as well as to cure traumatic and degenerative lesions of the knee joint. By an early and causal therapy, the new method according to the present invention allows to stop the progression of traumatic or degenerative joint diseases. For the treatment of osteoarthritis in young people, a unique and new therapeutic method is available with the present invention, which considerably delays the endoprosthetic joint replacement and in some cases avoids it. As an application of the method of the present invention it is also possible to treat the deterioration of the joints caused by inflammation.

The development of the gelatinous biomatrix of the present invention, in which autologous cells 20 can be included in exactly defined manner, allows for the first time to graft a defined amount of cartilaginous cells into a mechanically moldable and stress-resistant matrix. The cell-matrix-cartilage system of the present invention, constituted by the biomatrix according to the present invention and cartilaginous cells, can be prepared in any size and thickness and exactly adapted to the defect by mechanical adjustment. In comparison with the ACT, the biograft of the present invention also offers the advantage of suppressing further extraction of perioste from the patient's tibia. The in vitro culture of the cell-matrix-cartilage system of the present invention allows the ability of cartilaginous cells to produce matrix to be analyzed before transplantation. This is an important requirement to make cartilaginous grafts, check their functionality and ensure quality. Thus, it was possible to demonstrate that the new synthesis of typically cartilaginous proteins - such as type II collagen - that provide the fundamental stability of the graft is produced in vitro in the matrix by means of autologous cartilaginous cells. In accordance with the present invention, by tests on knee joints of Vietnamese pigs it could be demonstrated that, in comparison to the empty defect grafted with empty matrix without cartilaginous cells or with suspensions of cartilaginous cells according to the Peterson method, the cell systems Matrix-cartilage of the present invention is implanted excellently in the cartilage of the joint, completely fills the defect and also resists pressure. 35

Finally, the present invention also relates to a biomatrix for use in one of the aforementioned procedures.

The present invention also relates to methods for treating diseases or defects of the cartilage, especially degenerative, inflammatory and / or traumatic diseases of the joints, by using a cartilaginous prosthesis or a biograft prepared according to the present invention and / or of cultured cartilaginous cells 40 according to the present invention, to implant in tissues or cartilage - or in bone areas - affected or damaged or replace the affected or defective parts.

The present invention is illustrated in more detail by way of examples.

Example 1: Collagen Preparation

To prepare a collagen solution, a tissue containing collagen is used, such as for example 45 tendons of rat tails. All operations are carried out with sterile materials and under sterile conditions. To do this, after keeping them at -20 ° C, the rat tails are superficially disinfected for 5 minutes in 70% alcohol. The skin is then removed from the rat tails and the loose collagen fibers are removed. If another starting tissue is used, eventually the cells present can be carefully removed by mechanical, enzymatic or chemical treatment. The collagen fibers are collected in a phosphate buffered saline solution (PBS) (pH 7.2), 50 are superficially disinfected for 10 minutes in 70% alcohol and then washed with PBS. The weight of the collagen fibers is determined and they are transferred to a 0.1% acetic acid solution (approximately 8 to 12 mg / ml). The preparation is stirred for a period of 3 to 14 days and then the undissolved collagen particles are centrifuged (1000 rpm, 1 hour, at 8 ° C). In a preferred embodiment, the finished collagen solution has a collagen content between 3 mg / ml and 8 mg / ml. Therefore, as defined above, collagen is present in large proportion as a starting material in solution and not in the form of fibers, lattice or matrix. The resulting collagen solution is preferably cell-free and prepared from fresh, non-lyophilized collagen.

Example 2: preparation of the biomatrix or biograft

All operations are carried out with sterile materials and under sterile conditions.

Collagen solution: minimum 3 mg / ml in acetic acid

Buffer solution: 77.5 ml of doubly concentrated medium (for example DMEM)

20 ml of serum 5

2.5 ml of Hepes solution (3 M, pH 7.8)

Both solutions are stored at 4 ° C.

The biomatrix is formed by a 0.1% solution of collagen in acetic acid (rat tail collagen, with a collagen content of 3 mg / ml to 8 mg / ml, preferably 6 mg / ml) and a buffer solution of doubly concentrated medium, serum and Hepes solution. Shortly after mixing both components, preferably 10 in a 1: 1 ratio, a three-dimensional collagen structure gels at temperatures above 4 ° C, for example at room temperature.

To prepare a biograft, that is to say a biomatrix containing cells, 2 x 104 to 2 x 107 cartilaginous cells / ml are introduced - preferably 2 x 106, precultured in the usual manner and separated by centrifugation (1000 rpm, 10 minutes, at temperature ambient) - in the buffer solution, they are suspended and mixed at 4 ° C with equal parts of the collagen solution. This gel solution is pipetted into culture vessels and after gelling at 37 ° C forming a newly formed collagen structure, with cartilaginous cells included, it is coated with medium. The material is ready to transplant after 3 to 8 days of culture.

If desired, the cells can be separated again and obtained from the biomatrix by a collagen treatment followed by centrifugation. The resulting differentiated cells may be the starting point for another 20 cultures.

Example 3: graft in the open joint and arthroscopic application

Both during transplantation in the open joint and in the arthroscopic application, the cell-matrix cartilage first adapts mechanically in shape and size to the cartilaginous defect. Next, the graft is inserted into the crack and fixed there by fibrin adhesive. As the cell-matrix-cartilage system is a flexible but at the same time dimensionally stable material, this biograft is wound almost on tubular arthroscopic instruments and thus introduced into the crack. Thanks to its dimensional stability, the grafts recover their original shape at the intended site and can be fixed to the defect with fibrin adhesive.

Example 4: PCR test results

The following table reproduces the results of the PCR assays. The purpose of these tests was to demonstrate that the culture according to the present invention in a biomatrix prepared according to examples 1 and 2 of the present invention results in redifferentiation of the chondrocytes. In the table it can be seen that, starting from a P0 culture, chondrocytes lose their ability to produce type II collagen during successive conventional passages P1 and P2. The cells dedifferentiate in the course of these passages. In contrast, a passage P2 made in a biomatrix of the present invention results in the redifferentiation of the dedifferentiated chondrocytes, which is confirmed by the recovery of the ability to generate type II collagen.

Table: PCR test results:

RNA was isolated from the chondrocytes of each culture and transcribed into cDNA.

 P0-2D 3 weeks P1-2D 2 weeks P2-2D 3 weeks P2-collagen 2 x 106 / ml 3 weeks

 β-2-microglobulin

 + + + +

 Type I collagen

 + + + +

 Type II collagen

 + - - +

 Type XI collagen

 + + + +

 Bmp 2

 + + + +

 Bmp 4

 + + + +

 BMP 7

 - - - -

The expression (transcription) of the specific chondrocyte genes was assayed by PCR amplification with suitable primers and subsequent gel electrophoresis:

- β-2-microglobulin is a constitutively expressed gene and serves as a positive control;

- Type II and XI collagen are types of cartilage-specific collagen; 5

- Bone morphogenetic proteins (Bmp) 2, 4 and 7 play a role in the differentiation of cartilaginous tissue.

Claims (5)

1. Method of preparing a collagen biomatrix, which comprises the following steps: prepare collagen fibers from rat tails; transfer the collagen fibers to an acetic acid solution with a pH value of 3.0 to 4.0; incubate the collagen fibers in the acetic acid solution at a temperature of 2 to 10 ° C for a period of 3 to 14 days; separate the undissolved collagen particles, whereby a collagen solution is obtained whose collagen content is 3 to 8 mg / ml; Y Mix the collagen solution obtained with a buffer solution of 7.5 to 8.5 pH at a temperature of 2 ° C to 10 ° C and increase the temperature to achieve gelation, thereby obtaining a biomatrix with 10 1.5 until 4.0 mg of collagen per 1 ml of biomatrix and a pH value of 7.0 to 7.8. 2. A method according to claim 1, wherein the content of collagen fibers in the acetic acid solution is 8 to 12 mg per 1 ml of the acetic acid solution. 3. Method according to claim 1 or 2, wherein the concentration of the acetic acid solution is 0.1%. 4. Collagen solution prepared by the method according to one of claims 1 to 3. 15 5. Biomatrix prepared by the method according to one of claims 1 to 4.